We have previously shown that human folate binding proteins, or human folate receptors (hFR), are constituents of gene family of ubiquitously expressed glycoproteins (HFR-KB, hFR-P, and hFR) that mediate folate and antifolate transport. Our recent studies have demonstrated that expression of the hFRs is an important determinant of antifolate mediated cytotoxicity and of acquired drug resistance, and permits cellular proliferation of non-expressing cells under folate limiting conditions. Although the level of hFR expression is inversely related to folate concentrations and is highly variable among human normal and malignant tissue, the mechanisms regulating hFR expression are unknown. Furthermore, despite knowledge of the primary structures of hFRs, the 2 secondary and 3 tertiary structures and functional domains of the hFRs are undefined. In the past year, we completed the characterization of the hFR-KB gene, the structural and functional analysis of the P4 promoter of the hFR-KB gene, and the investigation of the role of conserved tryptophan residues in the structure and function of the hFR. In preliminary work, we are investigating the structure and function of the PI promoter of the hFR gene; the mechanism whereby folates affect hFR transcription; the role of methylation of the hFR genes on gene transcription; the normal tissue distribution of hFR cDNA isoforms; the mechanisms involved in tissue specific expression of hFR genes and isoforms; the effect of hFR antisense constructs on cellular proliferation; the role of glycosylation of the hFR on the functional properties of the protein; the sorting of the hFR with emphasis on the role of different membrane anchoring mechanisms (e.g. GPI vs. non-GPI membrane attachment); the physiologic requirements for folates independent of synthesis of DNA precursors; the role of folates (and antifolates) on neural tube defects in Xenopus laevis embryos. In general, the experimental Hematology Section is investigating a) the structure, function, and molecular biology of the hFRs, b) the role of hFRs in the transport of folates and antifolates and the mechanism whereby hFRs internalize ligand, and c) the regulation of hFR expression.